Introduction

Food/Techniques/Modelsystems
Niewold
 Nutrition
• Introduction

• Fysiology

• Complexity

• Modelsystems

• Conclusions
 Introduction

• Nobody can do without food

• Food is the dominant environmental

factor for gene expression

• Determinant for health

 Sources

• (Nutritional) Epidemiology
– determines associations nutrition and
health

• (Animal) models
Trends and technologies in nutrition science
Personalized
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THE AMERICAN DIETETIC ASSOCIATION (2003) , S50-55

 Fysiology
computing
Fysiologie

oesophagus
gall bladder

duodenum
h
liver ac pancreas
om
st
colon

colon
ileum

caecum

appendix rectum

Presentation copyright © 2002 David A Bender and some images copyright © 2002 Taylor & Francis Ltd 
 computing
salivary amylase
The gastro-intestinal tract -
lingual lipase
2

pancreatic amylase
lipase, phospholipase
trypsin, chymotrypsin, elastin
dipeptidases
gastric acid disaccharidases
pepsin
gastric lipase absorption of:
alcohol absorption monosaccharides
amino acids
fatty acids, glycerol, fats
water
bacterial fermentation
absorption of water

Presentation copyright © 2002 David A Bender and some images copyright © 2002 Taylor & Francis Ltd 
 computing
The gastro-intestinal tract -
bacteria aeroob
2

bacteria microaeroob
bacteria acid resistent anaeroob

bacteria anaeroob

Presentation copyright © 2002 David A Bender and some images copyright © 2002 Taylor & Francis Ltd 
 computing
The intestinal mucosa - 1

villi

muscle

Presentation copyright © 2002 David A Bender and some images copyright © 2002 Taylor & Francis Ltd 
 computing
The intestinal mucosa - 2

villi

muscle

Presentation copyright © 2002 David A Bender and some images copyright © 2002 Taylor & Francis Ltd 
 cells shed at tip of villuscomputing

Intestinal villi
absorptive
enterocyte

mucus secreting
villi goblet cell

lacteal

cell proliferation in crypt

muscle

lymphatic drainage
venous drainage
arterial blood supply

Presentation copyright © 2002 David A Bender and some images copyright © 2002 Taylor & Francis Ltd 
 Epithelium: Structure, tight junctions

para- transcellulair
 The intestines: a complex and dynamic
ecosystem

FOOD

Health
genetics
epigenetics ca 90%
immunology unknown
nervous MUCOSA MICROBIOTA
system
Mucosa
Commensal biota Pathogen

Lumen

Mucus

Epithelium

PMN Macrophage
B-cell Lamina
Cytokines: propria
Nutrients TNF
IL-1
T-cell IL-6 catabolism
appetite down
 Modelsystems 1

• Human: biopsy, fistels, stoma, faeces

etc?

• In vivo animal models

– small rodents/other species

– species specific
– pig<>human
 Sampling
computing
Fysiologie

oesophagus
gall bladder

duodenum, peroraal scope

h
liver ac pancreas
om
st

ileostoma
colon

colon
colostoma ileum
coloscope
caecum

appendix rectum

Presentation copyright © 2002 David A Bender and some images copyright © 2002 Taylor & Francis Ltd 
 Modelsystems 2

• In vitro cell systems

– monolayer
– Boyden chamber
– Ussing chamber

• In vitro cell free systems

– TIM
– SHIME
 Modelsystems 3

• In situ systems
– ligated loop
– SISP

• In vivo techniques
– fistulation
Monolayer

• Cell lines eg Caco-2

• Adhesion bacteria

• Gene expression

• Food components

• etc.
Monolayer caveats
• Cell lines eg Caco-2
microscopic
morphologically perfect,
functionally too?

• Aerobe

• Gene expression limited

• Misses interaction other

components
Mucosa
Boyden chamber
• As monolayer

• + polarity

• Transepithelial
electrical resistance

• permeation
– transcellular
– paracellular

• etc
Boyden chamber caveats

• As monolayer

• Co-cultures
Mucosa
Ussing chamber

Gasses
oxygen,
carbondioxide
Ussing chamber
example

• Transport
increases
permeabiliteit

• Implications
for toxins,
bacteria
 Ussing chamber

villi

muscle
 Ussing chamber 2

• Mucosa or
monolayer

• Permeation

• Electrophysiolo
gy

• max 4h mucosa
 Ussing with mucosa

FOOD
No circulation

genetics
epigenetics
immunology
nervous MUCOSA MICROBIOTA
system: none
 In vitro simulation of microbiota

• Advantages
– universal standard
– no ethical problems
– fast screening
– low cost
– mechanistic studies
• Limitations
– extrapolation to vivo?
– relevance for long term processes?

Labo Microb Ecol Technieken UGent

 TIM
(TNO gastric small Intestinal Model)
• Temp, peristalsis, transit

• stomach, intestines

• acid, gall, pepsin, lipase

• microbiota

• absorption: digests, water

• computer control
 Simulator of Human Intestinal Microbial
Ecosystem (SHIME)
pH pH
A P pH

Food Effluent
IV V VI
I II III

r r r r r r
A: Acid
I: Maag IV: Caecum/Colon ascendans P: Pancreas
II: Duodenum V: Colon transversum pH: pH-control
III: Jejenum/ileum VI: Colon descendens r: Stirrer

Labo Microb Ecol Technieken UGent

 Start of reactor

• Inoculation with fecal slurry

• Stabilisation of biota
– food
– pancreatic juice
– RT=65 h
– pH=5.6-6.9
– anaerobic conditions
– temperature (37°C)

Labo Microb Ecol Technieken UGent

 Control and sampling

• Bacterial composition: plating, DGGE, FISH, realtime

PCR, Flow Cytometer, Life/Dead
• Enzyme activity
• Fermentation pattern: fatty acids,
ammonium, gasses,
Metabolites

• Analytic, biological techniques

Labo Microb Ecol Technieken UGent
 Application
• Screening
– functional foods: pre-, pro-, synbiotica
– digestibility food
• (In)activation of bio-active molecules
– pharmaca, oestrogens, carcinogens, pollutants
• Bacterial survival, fage therapy
• Transfer genes

Labo Microb Ecol Technieken UGent

 In vitro simulation

FOOD

X Representative?
90% unknown

MUCOSA
X MICROBIOTA
Obligatory
epicellular bact?
 Ligated loop

• Segment of intestine, ligated, in situ

• Injection materials, e.g. toxins,

bacteria, food

• Swelling, bacterial replication,

adhesion etc
Ligated loop example

After injection of enterotoxin volume

length ratio increases
 Ligated loop

FOOD +
unphysiological
pressure,
peristalsis?
genetics
epigenetics
immunology
nervous MUCOSA MICROBIOTA
system: none
 SISP (Small Intestinal Segment Perfusion)
Small Intestinal Segment Perfusion (SISP)

• weaned piglets
• anaesthesia
• 10 segments (20
cm)

• Pathogens, toxins
• Antibacterials,
pharmaceuticals,
probiotics
• isogenic

• Net absorption
• Bacterial
replication/adhere
SISP
 SISP example

• Effects components 750

– on absorption
500
– on bacterial adhesion

net absorption
– etc 250

-250
mock ETEC
 Cystic fibrose/enterotoxin:
background
• Jejunum
• CFTR: Cl- channel
• Cl- / HCO3-
• enterotoxin > cAMP
• Cystic Fibrose
 secr Cl- ,abs H2O
 Normal Enterotoxin

H2O H+ <HCO3- >HCO3- H2O

Na+ Na+
Cl- Na+ HCO3- Na+ HCO3-

H+ Cl- H+
Cl - Cl-
Cl-
HCO3- HCO3-

cAMP
 Normal Cystic fibrose X

H2O H+ <HCO3- H+ <HCO3- H2O

Na+ NaCl <Na+
Cl- Na+ HCO3- Na+ HCO3- <Cl-

X
H+ Cl- H+ Cl-
Cl
-
Cl -

HCO3- HCO3-
 Enterotoxin+DPC Cystic fibrose X

H2O <HCO3- H+ <HCO3- H2O

Na+ NaCl <Na+
Cl- Na+ HCO3- Na+ HCO3- <Cl-

X
H+ Cl- H+ Cl-
Cl- Cl-
HCO3- HCO3-

cAMP
 SISP DPC +/- toxine
Netto vloeistof absorptie

1000 *
800
600

*** *
 l/cm2

400
200
0
-200
-400
-600
0- 0 8 16 32 64 128 256 4- 16-
DPC conc.
To make it even more
complicated
 SISP absorption Food Comp
A vs B
Product A & Absorption
750

500
nett abs

250

-250
B A x y F4 C
 SISP: E.coli F4 (PWD) and probiotic
bacteria

• E. coli F4 reduces
SISP
EFFECT OF PROBIOTIC BACTERIA the intestinal
1000
absorption
nett absorption

750
(L/cm )
2

500 • Probiotic bacteria

250
reduce this effect
0
control E.coli E.coli + probiotic
probiotic
 INTESTINAL FUNCTION:
ISCHAEMIA REPERFUSION SISP (IR-SISP)

• Manipulation of intestinal blood

supply, mimicking exercise, resting,
transport
• Influence of pathogens
• Parameters:
– absorption, pathogens, intestinal acidosis
(tonometry), intestinal permeability in
Ussing chamber
IR-SISP

pH

1 hour clamping of arterial

blood flow (e.g. 90%) leads to
intestinal acidosis
 INTESTINAL FUNCTION: USSING
CHAMBER using intact mucosa

• Ussing chamber: measurement of

epithelial integrity (electrophysiology,
permeability for macromolecules)

• 1. With IR-SISP
• 2. As ex-vivo model
 USSING CHAMBER: after IR-
SISP with food-additive
• Modulation of
MODULATION OF
PERMEABILITY intestinal
2 permeability
0.008
PERMEABILITY

1 0.05
• blocking of
translocation
0
• delivery of
control ischemia Comp X
compounds
 SISP

FOOD

Health?
genetics
Parameters
epigenetics
immunology
nervous MUCOSA MICROBIOTA
system
Fistulation

Sampling
anaeroob?
 Animal models microbiota

• Conventional

• High health status (Minimal disease)

• SPF (Specific Pathogen Free)

• Gnotobiont

• Germ free
 SPF

• In overpressure stables, barrier, filters,

humanised microbiota

• SPF: List which germs absent

• Not absolute, and what are pathogens?

• Eg: if rotavirus is persistent, list adapted

 Germ free
• From caesarean section

• In isolator (size limitations)

Germ
free=
CD/CD:

Caesaria
n derived
Colostru
m
deprived
Germ
free
 Germ free

• Abnormal mucosa

• Abnormal immune system

• Vertical transmission? Retrovirus?

 Gnotobiont

• From germ free

• In isolator

• Addition known microbiota

– monoassociation
– or more complex
 Germ free

FOOD

genetics
epigenetics
X
immunology?

X X
nervous
system MUCOSA MICROBIOTA
 Gnotobiont

FOOD

genetics
epigenetics
immunology? very
nervous limited
system MICROBIOTA
MUCOSA
 Compilation in vitro techniques

Mucosa Microbiot Food Function

a

Monolayer 1 cell aerobe component cell function

type s adhesion
Boyden 1 cell aerobe component + permeation
chamber type s

Ussing mucosa aerobe component +electrophysiolo

chamber s gy

TIM/SHIME none aerobe, everything digestion,

anaerobe fermentation
 Compilation in/ex vivo
techniques
Mucosa Microbiota Food Functions/Parameter
s
Individual intact (an)aerob all limites sampling
e uptake blood/urine

Ligated no (an)aerob all sampling

loop blood, e bacteri-
nerve growth/adhesion
swelling, no transit
SISP operatio (an)aerob all sampling
n stress e bacteri-
growth/adhesion
peristalsis, transit
 Conclusions

• Modelling difficult

• Each model has limitations

• Extrapolation with great caution

• Combine with other systems

Establishment of intestinal crypt
culture system from stem cells.

T Sato et al. Nature 000, 1-4 (2009) doi:10.1038/nature07935

Trends and technologies in nutrition science
Personalized
diets

Functie ?

THE AMERICAN DIETETIC ASSOCIATION (2003) , S50-55